Establishing the molecular basis of how neurons transmit signals is essential for a molecular understanding of learning, behavior, and memory. This knowledge will ultimately be essential for the design and improvement of therapeutic agents needed for treatment of psychological and neurological disorders. Neurotransmitter release into the synapse is mediated by membranous vesicles that dock and fuse with the synaptic membrane. The molecular machinery responsible for this process is common to all eukaryotic cells. Association of the SNARE proteins present on vesicle and target membranes is required for membrane fusion. The interaction of SNAREs is therefore strictly regulated in order to prevent inappropriate fusion events. In this proposal, a combination of biochemical, biophysical, and structural methods will be used to investigate the molecular mechanisms that govern SNARE assembly, and how SNARE complexes are disassembled after fusion. 1. nSec1/Munc18-1 is a member of the SM family of proteins required for vesicle trafficking and exocytosis. Munc18-1 binds with high affinity to the synaptic membrane SNARE syntaxin 1 and prevents syntaxin from engaging in SNARE complex formation. The factors responsible for releasing this inhibition are not known. In this project, these factors will be purified from brain extracts and their mechanism of action investigated biochemically and structurally. 2. SNARE assembly is coordinated with vesicular targeting and tethering to specific membranes. These "upstream" events are mediated by the Rab GTPases and their effectors. In this project, we will study the interactions of the yeast protein Sro7 and its mammalian counterparts, Lgl and tomosyn, that appear to mediate the link between Rab function and SNARE assembly. 3. The ATPase NSF mediates disassembly of the SNARE complex after fusion, so that the components can be recycled for subsequent rounds effusion. The conformational changes produced by nucleotide hydrolysis by NSF and the related ATPase p97 will be investigated by small-angle x-ray scattering and x-ray crystallography.